Harpagophytum procumbens (Hp), commonly known as Devil’s Claw, has been found to be of benefit as an anti-inflammatory and analgesic treatment, particularly in the symptomatic relief of arthritis (Wegener, 1999). Tissue damage associated with arthritic conditions is contributed to by the over-expression of free radicals, particularly nitric oxide (NO), by phagocytic cells (Chapman, 1989). We have investigated the effects of two Hp preparations (extract and tincture) on the 1,1-diphenyl-2-picrilhydrazyl ( DPPH) radical, and on nitrite release from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and from the NO-donator, sodium nitroprusside (SNP).

Cells were suspended in Dulbecco’s Modified Eagle’s Medium, 10% foetal bovine serum, and 2 mM L-glutamine and incubated in 24-well plates (1x10⁶ cells/well) for 1hr at 37ºC, 5% CO₂. Hp treatment (0.001–1mg/ml) of cells consisted of either; (1) 1hr pre-treatment before being washed and activated with LPS (100µg/ml) for 20hr; (2) no pre-treatment and 20hr concurrent treatment with LPS; or (3) 1hr pre-treatment plus 20hr concurrent treatment with LPS. Post-treatment cell viability was assessed with trypan blue and nitrite measured at 550nm with Griess reagent. Values were expressed as percentage inhibition of nitrite released by control cells with effects of diluent controls deducted (table 1). In cell-free systems, Hp solutions were added to equal volumes of DPPH (0.06 mM) or SNP (5 mM) solutions for 30 and 150min, respectively. DPPH was measured at 515nm and nitrite determined with Griess reagent. DPPH and nitrite scavenging were calculated as a percentage of control. All experiments were done in triplicate and on 3 separate occasions.

Table 1. Inhibition of nitrite release from LPS-stimulated cells as a percentage of control .

Data presented as mean ± s.e.m.

The IC₅₀ for DPPH scavenging was 49.87µg/ml and 33.45µg/ml for Hp extract and tincture, respectively. Maximum scavenging capacities of NO from SNP was observed at 1mg/ml achieving 27.81±1.22 % (extract) and 20.57±2.52 % (tincture) inhibition. Both Hp preparations were able to scavenge the DPPH radical and nitrite donated from SNP. The direct scavenging ability of Hp on nitrite may be responsible for the reduction in extracellular nitrite measured from LPS-stimulated macrophages. Hp may also inhibit inducible nitric oxide synthase since 1 hour pre-treatment, and subsequent removal of Hp from cells before addition of LPS, was found to attenuate nitrite levels. In conclusion, Hp appears to be an antioxidant able to suppress cellular NO in vitro, most probably through its ability to scavenge free radicals, though enzymic inhibition may also be implicated.

Chapman, M, et al (1989) J Rheumatol. 16: 15-18.
Wegener, T. (1999) Wein Med Wsher 149: 254-7.